Plant-based production of recombinant proteins has emerged as an efficient and cost-effective alternative to microbial fermentation and mammalian cell culture systems. Chloroplasts harbor high plasmid copy numbers and can be stably transformed, making them efficient platforms for protein production. In the present study, we used green fluorescent protein (GFP) as a reporter to compare the three major chloroplast promoters (rrn, psbA, and rbcL) involved in protein production in Nicotiana tabacum cv. “Petit Havana.” Three chloroplast transformation vectors were constructed, each regulated by a different promoter, and the transformation was performed via biolistic particle bombardment. Transformants were selected based on spectinomycin resistance and were confirmed by PCR. Among the three promoters, psbA showed the highest transformation efficiency and protein expression levels. Reverse transcription quantitative PCR showed that the mRNA levels (relative to Actin) for psbA (218.21±19.64) were nearly twice that of rbcL (126.60±8.78), and five times that of rrn (43.27±1.57). This transcriptional hierarchy was also observed at protein level. Immunoblotting showed the GFP levels (relative to psbA) were: psbA (1.00), rbcL (0.87), and rrn (0.77), whereas quantification through ELISA revealed relative GFP concentrations of: 616.2±28.7 ng/g LFW for psbA, 510.3±32.4 ng/g LFW for rbcL, and 338.9±100.2 ng/g ng/g LFW for rrn. These quantitative results demonstrate the importance of promoter selection for efficient expression of recombinant proteins in chloroplasts and show that the psbA promoter is suitable for high-efficiency chloroplast expression systems, providing a foundation for advancing plant-based molecular farming.

Perilla is an oilseed crop cultivated in Korea since ancient times. Due to the high α-linolenic acid content in perilla, perilla seed oil can easily become rancid. α-Linolenic acid is synthesized by two enzymes, endoplasmic reticulum-localized Δ15 desaturase (FAD3) and chloroplast-localized Δ15 desaturase (FAD7) in vivo. In order to lower the α-linolenic acid content of the seed oil without disturbing plant growth, we tried to suppress the expression of only the FAD3 gene using RNA interference, whilst maintaining the expression of the FAD7 gene. Seventeen transgenic plants with herbicide (Basta™) resistance were obtained by Agrobacterium-mediated transformation using hypocotyls of perilla plants. The transgenic plants were firstly confirmed by treatment with 0.3% (v/v) Basta™ herbicide, and the expression of FAD3 was measured by Northern blot analysis. The α-linolenic acid content was 10-20%, 30-40%, and 60% in two, seven, and three of the twelve T₁ transgenic perilla plants which had enough seeds to be analyzed for fatty acid composition, respectively. Analysis of the fatty acid composition of T₂ progeny seeds from T₁ plants with the lowest α-linolenic acid content showed that the homozygous lines had 6-10% α-linolenic acid content and the heterozygous lines had 20-26% α-linolenic acid content. It is expected that the reduction in α-linolenic acid content in perilla seed oil will prevent rancidity and can be utilized for the production of high-value functional ingredients such as high γ-linolenic acid.